Genetic manipulation of key proteins involved in the autonomic regulation process is impossible in rabbit f-SANC. So, we have developed a method to stable culture adult rabbit SANC (c-SANC), have characterized their properties, and have successfully overexpressed proteins in c-SANC via adenovirus directed acute gene-transfer technique. On the first day of primary SANC culture, most of the cells tend to spread out and could stay alive for up to 8 days, a reasonable period which would allow us to introduce exogenous proteins into c-SANC. The cultured SANC can beat spontaneously at 34 0.5 oC, and the action potential (AP) firing rate stabilizes at a level of 1.35 +/- 0.02 Hz (n=803, 2 to 8 days into culture), which is significantly slower than f-SANC (2.79 +/- 0.04 Hz, n=203, p<0.001). c-SANC generate regular and rhythmic AP, global Ca2+ release transients and local Ca2+ releases (LCRs) just prior to the Ca2+ transients triggered by spontaneous AP. The major characteristics of AP, Ca2+ transients and LCRs in c-SANC are similar to those from f-SANC, with some difference correlated with the spontaneous AP firing rate. By immuno-staining, we detected essential proteins involved in autonomic regulation in c-SANC, including type 2 sarcoplasmic reticulum Ca2+ release channel, i.e. ryanodine receptors (RyR2), L-type Ca2+ channel, hyperpolarization-activated cyclic nucleotide-gated channel 4, phospholamban (PLB), Sarco/Endoplasmic Reticulum Ca2+-ATPase 2a and Sodium-Calcium exchanger. It is well documented that the peptide PKA inhibitor, PKI can dramatically reduce or stop the beating rate of f-SANC. We hypothesized that the relatively low beating rate of c-SANC compared to f-SANC, is possibly due to the down-regulated protein kinase A (PKA) signaling in the cultured cells. Acute stimulation of beta-adrenergic receptors with 1microM isoproterenol (ISO) accelerates the AP firing rate to a similar maximum in c-SANC (3.34 +/- 0.05 Hz, n=150) and in f-SANC (3.55 +/- 0.06 Hz, n=126). In addition, we observed that the phosphorylation level of RyR2, which is indexed by the fluorescence density of phosphorylated RyR2 at Ser2809 normalized by the cells total RyR2 fluorescence density, is substantially lower in c-SANC (1.32 +/- 0.06, n=47) than that in f-SANC (1.66 +/- 0.15, n=24, p<0.01). While acute ISO stimulation raises the RyR2 phosphorylaiton at Ser2809 to a similar level in both cell types, PKI treatment reduces the phosphorylation level. More specifically, we measured the phosphorylation level PLB at Ser16, a PKA specific site, indexed by the fluorescence density of phosphorylated PLB at Ser16 normalized by total PLB fluorescence density. The results obtained with ISO acute stimulation and PKI inhibition in both cultured and freshly isolated SANC, support the interpretation that PKA signaling is down-regulated in cultured SANC compared with freshly isolated SANC. What is the mechanism underlying the PKA down-regulation in cultured pacemaker cells? We chose to measure the protein expression level of type 2 regulator of G protein signaling (RGS2), which functions as a powerful negative regulator of pertussis toxin (PTX)-sensitive Gi signaling. As we expected, the protein level is dramatically lower in c-SANC (149.9 +/- 4.0, n=100) than in f-SANC (201.9 +/- 6.0, n=88, p<0.001). Again, ISO (1microM, 2 hours incubation) enhances the staining density of RGS2 and PKI completely blocks ISOs effect. Furthermore, over-expression of RGS2 via adenovirus directed acute gene-transfer technique partially rescues the spontaneous beating rate of cultured SANC from 1.35 +/- 0.05 Hz (n=91) to 1.86 +/- 0.05 Hz (n=50, p<0.001). Introducing the green fluorescent protein (GFP) into c-SANC via adenovirus directed acute gene-transfer technique, does not affect the cell beating rate, and there is no correlation between AP firing rate and GFP expression level. When we treated cultured SANC with 0.4g/ml PTX overnight, the spontaneous beating rate promoted to 2.38 +/- 0.11 Hz (n=45). Partial rescue of s-SANC beating rate by PTX or RGS2 overexpression suggests the involvement of up-regulated Gi- signaling pathway in cultured SANC. We also successfully overexpressed some Ca2+ regulatory proteins in c-SANC, including wild type and mutant Ca2+ / calmodulin-dependent kinase IIgammaC (a multifunctional Ca2+ dependent kinase), Ht31 (a peptide that binds the PKA regulatory subunit type II (RII) and competes with endogenous A-kinase anchoring protein (AKAP) for RII binding, thus interrupts AKAP mediated PKA anchoring) and its inactive form Ht31p. We concluded that adult c-SANC provide a reliable model to study the autonomic regulation by acute genetic manipulation of key proteins. Moreover, understanding the difference between freshly isolated and cultured SANC itself broaden our view of autonomic regulation.